General aspects: Currently, several strategies have been used to overcome common problems
found in pharmacological therapies, such as insolubility, reduced bioavailability
and low specificity of drugs. One of the most promising alternatives proposed is the
use of dendrimeric drug nanocarriers, polymeric chemical structures of diverse nature
that contain, transport and deliver the desired drug in biological systems. One of
the dendrimers that has been most successfully used is based on polyamidoamine (PAMAM).
PAMAM are organized from a central molecule of ethylenediamine that gives way to expansive
growing layers (generations) terminating in a surface of primary amines that are positively
charged at physiological pH. This architecture determines the presence of intramolecular
cavities that allow the encapsulation of drugs and their release (
Figure 1
). Alternatively, drugs can also be associated to the dendrimer surface. Among the
main advantages of PAMAM dendrimers is their high solubility, stability and efficient
encapsulation of different drugs, in addition to its easily modifiable surface. This
latter feature allows the linking of several chemical groups and molecules to the
surface amino groups in order to improve their properties, such as surface charges,
encapsulation capacity and drug delivery, ligand linkage to reach a specific target
tissue, among other applications (Svenson, 2009). The versatility of the PAMAM dendrimers
has demonstrated to be useful in studying the action of several drugs of high biomedical
impact. Thus, anticancer, anti-inflammatory and antimicrobial agents, among others,
have been tested with promising results (Svenson, 2009). However, the pharmaceutical
use of such systems in neuropathology is a field that is yet to be explored.
Figure 1
Schematic representation of the chemical structure of polyamidoamine (PAMAM) dendrimers.
The main details are highlighted: The blue squares show the surface amino groups protonated
at physiological pH and the ethylendiamine central core; the red circle indicates
the space where drugs could be encapsulated. The different generations of polymer
layers are marked in tones of grey.
Cell internalization: It is generally accepted that a major advantage for using dendrimers
as carriers is their ability to enter the loaded drug into the cell. Thus, they can
circumvent problems not only for insolubility or permeability of some drugs, but also
allow their distribution to intracellular targets. Indeed, one of the main issues
to investigate is their mechanism of action for entry into the cytoplasm (
Figure 2
). There is evidence suggesting that the composition, size and ionic charges of the
dendrimers are relevant not only for the internalization mechanism induced, but also
for the kinetics of endocytosis and for intracellular processing mechanisms of these
polymers. Using specific inhibitors for clathrin and caveolin mediated endocytosis
and micropinocytosis, it has been established that the composition of surface charges
of PAMAM affects the internalization pathway. The evidence shows that anionic dendrimers
are internalized by caveolin mediated process, while the cationic and neutral dendrimers
appear to be taken up by a caveolin and clathrin independent process in A549 cells
(Perumal et al., 2008). However, colocalization studies with specific endocytic pathway
markers in HeLa cells show that the cationic dendrimers are internalized by clathrin
mediated endocytosis and micropinocytosis (Albertazzi et al., 2010), which demonstrate
that the process is dependent of the cellular type, too. The chemical versatility
of PAMAM dendrimers allows the linkage of chemical groups to interact with plasma
membrane proteins inducing their endocytosis in specific tissues or cells. Even when
endocytosis appears to be the main mechanism of dendrimer internalization, it cannot
be discarded that passive diffusion could have a role in that process. To precisely
analyze all these points is of major importance in order to focus dendrimeric polymer
studies on clinical pharmacology applications.
Figure 2
Models that explain the intracellular delivery of drugs mediated by polyamidoamine
(PAMAM) dendrimers.
Dendrimers could be internalized into the cytoplasm by non specific endocytosis. As
an alternative, dendrimers could be functionalized to allow the specific interaction
with transmembrane proteins inducing their endocytosis. The intracellular delivery
of drugs could be mediated by diffusion from the internal spaces of the dendrimers,
or by their disorganization in the intracellular vesicles.
Cytotoxicity: One of the main aspects to be considered in relation to the use of nanotechnologies
applied in biological problems are the toxicity levels. It has been widely recognized
that the presence of positive charges, provided by amino groups on the surface of
PAMAM dendrimers, means increased cytotoxicity levels, which is also determined by
the polymer size and surface composition. On the contrary, it has been demonstrated
that dendrimers containing only neutral or anionic surface groups are less toxic (Lee
et al., 2005). In this regard, and like other nanopolymers, autophagy process would
have a major role in the overall cellular response to such molecules (Wang et al.,
2014). The properties of the plasma membrane of cells treated with PAMAM dendrimers
could be affected. In electrophysiological experiments, it was determined that PAMAM
G5 increased the influx of Na+ in hippocampal neurons by a mechanism that needs to
be studied further (Nyitrai et al., 2013b). Another important aspect related to this
issue is the biodegradability of these polymers. Indeed, PAMAM dendrimers are hydrolytically
degraded only under harsh conditions because of their stable amide backbone, and hydrolysis
proceeds slowly at physiological conditions (Lee et al., 2005). However, there is
no strong experimental evidence about the mechanisms by which nanoparticles are degraded
and eliminated. Enzymatic activity cannot be discarded considering that amide bonds
of PAMAM dendrimers are similar to the ones present in other biodegradable molecules
like proteins. To overcome toxicity problems and improve the properties of dendrimers
in general, it is possible to covalently modify their surface linking molecules to
the amino groups. A common modification is the addition of polyethyleneglycol molecules
(PEG) of different sizes with a demonstrated reduction in toxicity. Several research
groups have addressed these issues making it clear the great potential of “pegylated”
PAMAM in biomedical applications. Formulations based on pegylated PAMAM dendrimers
with have shown an increased anticancer drug encapsulation and bioavailability with
a reduction in cytotoxicity. These kinds of complexes were internalized by endocytic
pathways, delivering the drug inside lysosomal vesicles with a demonstrated tumor
accumulation (Zhu et al., 2010). Other chemical modifications have been used to decrease
the cytotoxicity of dendrimers. For example, PAMAM with 4-carbomethoxy pyrrolidone
surface groups (PAMAM-pyrrolidone) shows a significative decrease of cytotoxicity
in Chinese hamster fibroblasts (B14), embryonic mouse hippocampal cells (mHippoE-18)
and rat liver derived cells (BRL-3A). Moreover, this modification prevents the increase
of intracellular ROS levels and mitochondrial membrane potential alterations caused
by PAMAM in this cell lines (Janaszewska et al., 2013). In general, as describes above,
cytotoxicity of the positive charged amines is not a limitation since it could be
reduced easily with chemical modifications, making nanocarrier systems based on PAMAM
dendrimers a biocompatible and versatile tool.
Neurobiological applications: Nowadays, one of the most important global health problems
are neurological disorders which are a continuous challenge for pharmacological research
and companies. Over 1.5 billion people worldwide are suffering from the central nervous
system (CNS) diseases. They currently represent 11% of the global burden of disease,
which is expected to rise to 14% by 2020, becoming one of the largest and fastest
growing areas of unmet medical need. A crucial aspect to consider when directing drug
therapy specifically to the CNS is the ability to cross the blood brain barrier (BBB).
About 98% of small molecule drugs fail to cross this barrier, whereas no large molecule
drugs cross the BBB except for a few natural peptides and proteins, such as insulin
(del Burgo et al., 2014; Xu et al., 2014). It is possible to functionalize PAMAM dendrimers
with specific BBB transmembrane protein ligands that target the nanocarrier to the
endothelial cells and help the drug internalization through specific endocytic processes.
Even when a more detailed description about the mechanism of drug delivery is needed,
it is probable that cargo molecules would be released from the dendrimer into the
endothelial cells to reach the neuronal target. The main advances that have been reported
are in relation with the use of LRP-1 receptor, transferrin receptor, EGF receptor
and integrin receptors (del Burgo et al., 2014; Xu et al., 2014). For example, PAMAM
functionalized with peptides derived from Kunitz domains (Angiopep-2, specific for
LRP-1 receptor) are capable of targeting the brain tissue in mice models (Ke et al.,
2009). Moreover, T7 peptide functionalized dendrimers loaded with the antitumor gene
agent pORF-hTRAIL and doxorubicin target the transferrin receptor and induce a three
times survival increase in a mice brain glioma model (Liu et al., 2012), which is
a demonstration for the effectiveness of these new nanocarrier systems. For optimal
therapeutic application, it is important to employ dendrimeric polymer systems which
do not generate significant changes in the physiology of cells, and in particular
in plasma membrane physiology and function. In this case, it is not enough that these
compounds are not cytotoxic, is also important that properties such as the integrity
of the plasma membrane and permeability to ions are not altered. Indeed, there is
experimental evidence showing that PAMAM having five generations (G5) did not effect
the conductance of K+ and Cl– ions in hippocampal neurons, but produced an increased
influx of Na+ (Nyitrai et al., 2013b). While the authors attributed this phenomenon
to the formation of pores in the plasma membrane as a result of the interaction between
the positive charges of the dendrimers and the cell surface, the specificity that
this pore shows for Na+ ions is not fully explained. Moreover, it has been shown that
PAMAM G5 are capable of inducing a significant increase in intracellular Ca2+ leading
to mitochondrial depolarization in pyramidal neurons and astroglial cells, indicating
that the focus must be not only in neuronal cells, but also in glial cells. These
studies also show that astrocytes are more resistant than neurons to the cytotoxic
effects of dendrimers (Nyitrai et al., 2013a). Another important application of dendrimeric
polymer systems is the intervention of cells or tissues with genes or interfering
nucleic acids. This has the advantage of eliminating the use of viruses as carriers
for these kinds of molecules and confers specificity to the tissue or cells to be
transfected. For example, a recent study by Brunner et al. (2015) demonstrated a high
efficiency in neuronal gene silencing with a direct application to rabies infection,
a relevant biomedical problem. In this case, a siRNA was linked to the surface of
a dendrimeric polymer, and a covalently associated cannabinoid receptor ligand conferred
specificity to the neurons (Brunner et al., 2015). Interestingly, beyond the nanocarreir
properties of PAMAM dendrimers, it has been described to have neuropharmacological
activity by itself. In this way, studies demonstrate that these polymers are able
to prevent the Alzheimer's peptide Aβ1–28 and prion peptide PrP 185–208 aggregation,
processes associated to neurodegenerative diseases. The probable mechanism for this
PAMAM effect is that the dendrimers interact with peptide monomers, and therefore
inhibit their capability of growing into fibrils (Klajnert et al., 2007).
Perspectives: In recent years, nanoparticles of several kinds have been developed
in clinical trials or patents directed to relevant biomedical problems (Cheng et al.,
2015), which is a demonstration that neurological applications of PAMAM dendrimers
are a real and feasible alternative. One of the issues to be addressed regarding the
use of nanocarriers in central nervous system is related to the possibility to increase
the tissue specific drugs delivery. In this regard, the studies of dendrimers passing
through the BBB and the understanding of specific association with tissues or cell
groups are highly relevant. Therefore, the generation of nanocarriers with activity
in brain nuclei or specific nerve centers to increase the effectiveness and reduce
secondary effects in treatments against neurodegenerative disorders, psychiatric pathology
and addiction is an area for future possibilities. Another interesting aspect that
neuropharmacology should address is the contribution that nanocarriers could have
in behavioral pharmacological studies or higher neural functions, as a first step
towards a possible application to human neuropathology. To reach this goal, it is
necessary to focus on studies that describe aspects of nanocarrier activity at the
subcellular level. Of particular interest is to know the possible applications of
this kind of drug delivery system in the regulation of synaptic function (
Box 1
). It is also necessary to further analyze the effects that dendrimers might have
on the basal activity of different components in neurons such as ion channels, neurotransmitter
transporters, vesicle mobilization systems, among other relevant aspects for neurological
applications.
Box 1
Polyamidoamine (PAMAM) dendrimers: actual knowledge and future perspectives.